Detection of bacterial pathogen on corn plants and test the potential of endophytic microbes to controlling stewart wilt disease in vitro

The use of synthetic pesticides to increase maize production sustainably contributes to increasing the development of pathogens and tends to be less effective in environmental conditions that support pathogens. In addition, the emergence of environmental pollution due to the use of chemicals causes the need for environmentally-friendly control technologies. Endophytic microbes are biological agents that can be used to plant disease control that is environmentally friendly and practical in their application. The study aimed to detect pathogenic bacteria that cause wilt and blight in maize and then examine the potential of several endophytic microbial isolates in controlling Stewart wilt pathogens in maize in vitro. The research was carried out from February to July 2021 at the Cereal Crop Research Institute Laboratory in Maros for morphological identification and microbial potential testing, while serological tests were carried out at the Makassar Plant Quarantine Laboratory, South Sulawesi. The study began with detecting pathogenic bacteria serologically using the Enzyme-Linked Immunosorbent Assay (ELISA) method. The results showed that blight symptoms on corn isolated from the experimental garden of IP2TP Bajeng Gowa Regency PS-4 and PS-5 were detected as bacteria Pantoea stewartii subsp. stewartii. The antagonistic test of endophytic bacterial isolates I.A1, II.D1, III.A2, IV.B2, and endophytic fungi isolates AC-1, AC-3, and DC-5 was able to suppress the growth of pathogenic bacteria isolates PS-4 and PS-5 The growth suppression of pathogenic bacteria was observed by forming inhibitory zones of antibiosis compounds. Pathogenic bacteria Pantoea stewartii subsp. stewartii can be controlled using endophytic microbes.


Introduction
Corn is the main food after rice which is also a concern of the Government, especially the Ministry of Agriculture.Apart from being a staple food ingredient, corn is also a processed ingredient for cooking oil, flour, organic acids, the animal feed industry, ingredients for making milk and ice cream [1,2].Along with 1253 (2023) 012005 IOP Publishing doi:10.1088/1755-1315/1253/1/012005 2 the increasing demand for corn, the quality must be maintained: disease control.Some of the main diseases of maize in Indonesia can be caused by bacteria and fungi, including leaf blight caused by the fungus Bipolaris maydis, leaf rust by the fungus Puccinia sp., banded sheath leaf and blight by the fungus Rhizoctonia solani, wilt disease, and Stewart bacterial leaf blight by Pantoea stewartii subsp.stewartii and stem rot disease caused by the bacterium Dyckea zeae [3][4][5][6] So far, farmers have a habit and dependence on chemicals such as synthetic fertilizers and pesticides in managing their farming business.These inputs have contributed significantly to increasing crop productivity and quality over the last 100 years.However, the use of chemicals such as fertilizers and pesticides that are not appropriate in controlling plant diseases also contributes to increasing the development of pathogens, and even their application tends to be less effective in conditions of high inoculum or the varieties planted are very susceptible [7] In addition, the emergence of environmental pollution causes major changes in people's attitudes towards the use of synthetic pesticides [8].
Another approach used in disease control is using biological control agents that are antagonistic to pathogens, environmentally friendly, and easy to apply.One of the biological control agents that are antagonistic is endophytic microbes.Endophytic microbes are a group of microorganisms that colonize the internal and intercellular spaces of all plant parts but do not cause disease or major morphological changes in the host and can be antagonistic to pathogens.The use of endophytic microbes as biological control agents is very interesting because it is known that endophytic microbes can colonize tissues, produce antimicrobial compounds, and increase tolerance to biotic and abiotic stress [9][10][11].
Currently, biological control agents for plant disease control have been widely reported, including Bacillus subtilis, B. cereus, Trichoderma spp., and Gliocladium sp.[12][13][14][15].Considering that the ability of each endophytic microbe is very diverse, the search and identification of the ability of isolates to suppress the development of pathogens are still being carried out.
The current study was aimed to detect pathogenic bacteria that cause wilt and blight in maize plants and test the potential of several endophytic microbial isolates in controlling Stewart wilt (Pnss) pathogens in maize plants in vitro.

Materials and methods
The research was carried out at the Plant Disease Laboratory of the Indonesian Cereals Research Institute (ICERI) in Maros to identify morphology, physiology, and antibiosis testing of endophytic microbes.In contrast, serological tests were carried out at the Plant Quarantine Laboratory in Makassar, South Sulawesi.They were starting from February to July 2021.The isolates of endophytic and pathogenic microbes used are from the collection of the ICERI Diseases Laboratory.Endophytic microbial isolates were collected from healthy maize, stems, and leaves.

Preparation of Pnss extraction from corn leaves (TAS 2018)
Samples came from corn leaves that showed symptoms.The infected and uninfected parts were separated, cleaned on the surface, and dried with paper.Then cut the sample into small pieces (Ø 5 mm).Samples of leaves that have been cut are soaked in distilled water.Surface sterilization was done by soaking the sample in a 0.85% calcium chloride solution with an adjusted volume.

Pnss isolation and purification.
The solution prepared in step (a), which gives a positive result on the serological test (b), is diluted by serial dilutions ten times with water or 0.85% sodium chloride.100 μl of each dilution was spread over an NA medium in Petri dishes containing 5% sodium chloride.The Petri dish was incubated at 30 °C and observed after 3-4 days.Pnss colonies are yellow, shiny, flat or convex, translucent, and have a sunken surface.

Identification.
Pure cultures of Pnss bacteria were observed for biochemical characteristics such as Gram staining, catalase test, growth test on selective media [16] and hypersensitivity reaction test using tobacco plants.

Antibiotic test of endophytic microbe against pathogenic bacteria
Pnss.A total of 5 isolates of endophytic fungi were tested for their antibiosis against pathogenic bacteria.Antibiosis test using the dual culture method, both isolates of endophytic fungi and pathogenic bacteria were grown on the same medium, namely Potato Dextrose Agar (PDA), but the pathogenic bacteria were first distributed as much as 100 l.Furthermore, endophytic fungus isolates (Ø 5 mm) were planted in the center of the Petri, then incubated for 3-5 days.Endophytic fungi were grown on a medium without pathogenic bacteria as a control.

Results and discussion
Detection of bacteria that cause wilt disease in corn plants begins with sampling symptomatic plants in the field.Symptoms of the disease found in maize cultivation in the districts of Maros and Gowa varied, but it was clear that the lines of necrosis on the leaves of maize plants were different from those of healthy maize (Fig. 1).Disease symptoms in maize plants infected with the pathogen Pantoea stewartii subsp.stewartii (Pnss), of which there is leaf blight with a characteristic line of necrosis in the middle of the leaf and blight on the edges of the leaf with an irregular pattern [17].

Identification of morphology, physiological tests, and biochemistry
Morphological observations were carried out on five pure isolates isolated from symptomatic leaves.In NA medium, bacteria have growth that varies from very fast to very slow (Figure 2).PS-1, PS-2, and PS 5 isolates had fast colony growth within 1x24 hours, in contrast to PS-3 and PS-4 isolates, which tended to slow colony growth up to 3x24 hours.The isolated Pnss bacteria had a variety of colonies; round shape, white and yellow color, and convex elevation, two isolates were slimy, and the periphery of the colonies varied (Table 1).Pnss is characterized by yellow colonies on culture media [17].The results of the Gram test, catalase test, and growth on YDC media conducted on five pure isolates showed that two bacterial isolates belonged to the Gram-negative group, and three isolates belonged to the Gram-positive group of bacteria.These isolates could produce catalase enzymes, but on growth testing on YDC media, only PS-4 isolates showed a positive reaction to keep growing with yellow pigment (Table 1).

Table 1. Characterization of morphology and physiology of pathogenic bacterial isolate.
The hypersensitivity test (HR) on tobacco plants showed positive reactions only on PS-4 and PS-5 isolates, while the other three isolates showed negative reactions.A positive response was indicated by the presence of necrosis symptoms in the inoculated part of the pathogenic bacteria.According to [ 16] that the hypersensitivity response in plants is that the symptoms begin with the yellowing of tobacco leaves until there is limited necrosis of the tissue infiltrated by the pathogen suspension.

Endophytic fungal antibiosis test against Pnss. pathogenic bacteria
The antibiotic activity of endophytic fungi isolates against pathogenic bacteria of corn plant isolates PS-4 and PS-5 was indicated by the clear zone formed around the isolates of endophytic fungi ( Endophytic bacteria and fungi are known to produce secondary metabolites so that it is more likely to inhibit the growth of pathogens through an antibiosis mechanism [ 1 8 ] .In addition to the production of antibiotic compounds, endophytic microorganisms are also reported to produce protease and lipase enzymes that are thought to be related to their ability to suppress the growth of pathogens [19].The inhibitory effect of endophytic fungi isolates against PS-4 and PS-5 isolates is shown in Table 3, it was observed that the treatment of endophytic fungi had a significant effect on the growth of Pnss pathogenic bacteria isolates.The endophytic fungi isolate AC-1, AC-3, and DC-5 were observed to be significantly different from the control treatment with 32-38% suppression effectiveness against pathogenic bacteria Pnss PS-4 isolate.While the pathogenic bacteria Pnss PS-5 isolate was effectively inhibited by endophytic fungi isolates DC-5, AC-1, and AC-3 which were observed to be significantly different from the control treatment with suppression between 31-34%.The ability of endophytic fungus isolates to inhibit the growth rate of pathogenic bacteria Pnss PS-4 isolate was observed from the inhibition zone formed with a range between 0.8-3.1 cm (Figure 3).At 24-48 hours after inoculation, it was observed that there was no significant effect between the treatment of endophytic fungi and the suppression of the growth of Pnss pathogenic bacteria.However, observations 96 hours after inoculation showed that the diameter of the inhibition zone of isolate DC-5 was significantly higher than that of isolates AC-2 and AC-6 and not significantly different from isolates AC-1 and AC-3.Thus, endophytic fungi isolate AC-1, AC-3, and DC-5 were effective biological agents for the pathogenic bacteria Pnss isolates PS-4 in maize.In line with the research results of Yuan et al. [20], endophytic fungi isolate CEF-818 and CET-714 were able to reduce Verticillium wilt infection in cotton plants.The ability of endophytic fungi isolates to inhibit the growth rate of Pnss pathogens PS-5 isolates was observed from the clear zone formed with a range between 1.4 -2.8 cm (Figure 4).Of the five isolates of endophytic fungi tested, the diameter of the inhibition zone was not significantly different against the suppression of Pnss pathogenic bacteria in the observation period of 24 to 96 hours after inoculation, so it can be said that the endophytic fungal isolates AC-1, AC-2, AC-3, DC -5, and AC-6 have the potential to be used as biological agents for the pathogenic bacteria Pnss isolate PS-5.Note: the numbers followed by the same letter in the same column are not significantly different on the LSD test α= 0.05.

Endophytic bacteria antibiosis test against Pnss pathogenic bacteria
Antibiotic activity of endophytic bacteria against pathogenic bacteria isolates PS-4 and PS-5 was indicated by the inhibition zone formed around the filter paper that had been dripped with endophytic bacteria (Figure 5).Endophytic bacteria are potential biocontrol agents against vascular wilt disease because they have a similar bioecology to the pathogens that cause vascular wilt [21].Observations on the inhibition of pathogenic bacteria were carried out by growing isolates of pathogens and endophytic bacteria in one TSA medium in a Petri dish.The effectiveness of suppressing the growth of pathogenic bacteria Pnss PS-4 and PS-5 isolates by 25 isolates of endophytic bacteria is shown in Table 4.The results show that the treatment of endophytic bacteria significantly affected the effectiveness of inhibition of isolates of pathogenic Pnss bacteria.The best suppression was observed in the endophytic bacterial isolates II.D4.1 against the pathogenic bacteria Pnss isolates PS-4 significantly different from the positive control treatment with an effectiveness of 67%, while the endophytic bacteria II.D2, II.D4. 1, and III.A1 was observed to be significantly different from the control treatment with the highest inhibitory effectiveness against pathogenic bacteria Pnss isolate PS-5 ranging from 7375%.All isolates of endophytic bacteria grown on NA media were able to provide an inhibitory zone against Pnss bacteria with high inhibitory capacity varies.Endophytic bacteria have various abilities to suppress the growth of pathogenic bacteria that cause the vascular wilt of corn plants.One of the mechanisms of suppressing endophytic bacteria against pathogens is the production of antibiotic compounds [22].Note: the numbers followed by the same letter in the same column are not significantly different on the LSD test α= 0.05.
Isolates of pathogenic bacteria PS-4 and PS-5 are thought to have contributed to the ability of endophytic bacteria to suppress the growth of Pnss, this can be seen from several isolates of endophytic bacteria whose inhibitory power on isolates of pathogenic PS-4 was not as good as that of isolates PS-5 and vice versa.

Figure 1 .
Figure 1.Samples of corn plants suspected of being infected with pathogenic bacteria Pantoea stewartii subsp.Stewartia.

Figure 2 .
Figure 2. Morphology of pathogenic bacteria isolates on TSA medium.

Figure 3 .
Figure 3.The ability of endophytic fungi to inhibit the growth of Pnss bacteria isolates PS-4 at 24, 48, and 96 hours after incubation.Note: the numbers followed by the same letter in the same column are not significantly different on the LSD test α= 0.05.

Figure 4 .
Figure 4.The ability of endophytic fungi to inhibit the growth of Pnss bacteria isolates PS-4 at 24, 48, and 96 hours after incubation.

Table 2 .
Antibiotic test results of endophytic fungi against pathogenic bacteria in vitro 3 days after inoculation (DAI).

Table 3 .
Inhibition effectiveness of endophytic fungal isolates on the growth of Pnss pathogenic bacteria isolates observed at 96 hours after incubation.Note: the numbers followed by the same letter in the same column are not significantly different on the LSD test α= 0.05.

Table 4 .
Inhibition effectiveness of endophytic fungal isolates on the growth of Pnss pathogenic bacteria isolates observed at 48 hours after incubation.